A. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly it relates to latent TGF.beta. binding protein (LTBP) genes, compositions and methods of use.
B. Description of the Related Art
1. TGF-.beta.
Five TGF-.beta. family members, which share 66-82% sequence identity, have been identified (Kingsley, 1994). Whereas TGF-.beta.1 was cloned from a cDNA library derived from human placenta, TGF-.beta.2 was subsequently purified from several mammalian cells and tissues, and TGF-.beta.3, -.beta.4, and .beta.5 were cloned by low stringency hybridization from mammalian, avian and amphibian cDNA libraries, respectively. Peptide growth factors/cytokines have also been identified that share sequence homology (.ltoreq.40%) with the TGF-.beta.s (collectively, the TGF-.beta.s plus these other cytokines make up the TGF-.beta. superfamily). A unifying feature of the biology of these other cytokines (i.e., the Mullerian inhibiting substance, bone morphogenetic proteins, growth and differentiation factors, activin/inhibin, Drosophila decapentaplegic complex, and amphibian Vg1 protein) is the ability to regulate developmental processes. In every case where information is available, superfamily members are synthesized as larger precursors that are processed at endoproteolytic cleavage motifs, and they terminate with the sequence C-X-C-X. The three dimensional crystal structure of the TGF-.beta.2 homodimer was recently reported (McDonald and Hendrickson, 1993). This work has led to the interesting and novel suggestion that TGF-.beta. is related to certain peptide growth factors (e.g., NGF, PDGF, v-SIS) in a way that could not have been predicted from the deduced amino acid analysis.
2. Latent TGF-.beta. Complexes
Many cell types produce TGF-.beta., and almost all cells bind TGF-.beta. with affinities in the picomolar range--e.g., the type I and type II TGF-.beta. cell surface receptors (glycoproteins of 53 and 75 kDa, respectively) are present in essentially all cells (Miyazono et al., 1994). Thus, TGF-.beta. has powerful effects on most cell types, and cytokines such as TGF-.beta. are thought to exert broad control the tissue remodeling that occurs during development, wound repair, and other situations (Sporn et al., 1986; Moses et al., 1990). (For a comprehensive review of TGF-.beta. effects, see Roberts and Spom, 1990). For example, TGF-.beta. was initially identified as a factor that stimulated the anchorage independent growth of rodent fibroblasts (Assoian et al., 1983; Frolik et al., 1983; Roberts et al., 1983). It is now known, however, that TGF-.beta. acts as a potential growth inhibitor for most cells, i.e., epithelial, endothelial, and hematopoietic progenitor cells; both stimulates and inhibits cellular differentiation; induces extracellular matrix production by stimulating the expression of matrix macromolecules, stimulating the expression of matrix protease inhibitors, and decreasing the expression of matrix degrading proteases; inhibits the functional activities of immune cells; induces the chemotaxis of fibroblasts, macrophages, and smooth muscle cells; induces angiogenesis in vivo; inhibits endothelial migration; induces the expression of cell surface receptors for other cytokines (e.g., the EGF receptor); promotes the healing of incisional wounds; inhibits osteoblast proliferation in vitro; and induces new bone formation in vivo.
A molecular explanation for these complex (and, at times, conflicting) effects is not yet available, but hypotheses do exist. Sporn et al. (1986) have suggested, for example, that the ability of TGF-.beta. to stimulate or inhibit the proliferation of mesenchymal cells depends on the state of cellular differentiation and the entire set of growth factors operant in that cell population. As such, the "biological meaning" of TGF-.beta. signal transduction depends on the context (i.e., availability and presentation) of other growth factors present in the local environment: Fischer rat 3T3 cells transfected with a myc gene and incubated with TGF-.beta. and PDGF proliferate in soft agar, whereas the same cells in the presence of TGF-.beta. and EGF fail to grow (Roberts et al., 1985).
Whatever the mechanism, the autocrine and paracrine activities of TGF-.beta. clearly must be regulated with precision. One regulatory strategy involves the temporal and spatial control of TGF-.beta. gene expression. A second strategy involves the production and storage of TGF-.beta. as a latent complex that is activated only under certain physiological and pathological conditions--e.g., tissue morphogenesis and remodeling, and wound healing. TGF-.beta.1 can be isolated from serum and from most tissues as a latent complex (Pircher et al., 1986; Miyazono et al., 1988; Wakefield et al., 1988). In this regard, the latent complex has been purified from human platelets and characterized in detail (Miyazono et al., 1988). Following a 6-step protocol, the purified complex yielded protein bands of Mr 25,000 and 210,000 on SDS-PAGE under nonreducing conditions. After reduction, the 25 kDa band was shown to consist of subunits of Mr 12,500. On the other hand, the 210 kDa band consisted of a Mr 40,000 subunit and Mr 125-160,000 subunit.
TGF-.beta. is also secreted from several producer cell lines in culture as a latent complex of 235 kDa (Gentry et al., 1987). TGF-.beta.1 is initially synthesized in vitro as a 390 amino acid precursor that consists of a signal peptide, an amino-terminal propeptide, and the mature growth factor. Two precursor chains associate to form a disulfide-bonded dimer with latent activity. Homodimers occur most commonly, but heterodimers may also form (Ogawa et al., 1992). The full length dimer is cleaved at a endoproteolytic cleavage motif, but the propeptide dimer (i.e., the latency associated peptide or LAP) and the mature growth factor dimer typically remain non-covalently associated. The mature TGF-.beta. dimer is now known to be the 25 kDa band identified after nonreducing SDS-PAGE of the purified latent complex from platelets. In addition, LAP is known to be a component of the 210 kDa band identified after nonreducing SDS-PAGE of the purified latent complex from platelets (i.e., LAP has been shown to consist of two of the 40 kDa subunits).
Together, LAP and the mature TGF-.beta. dimer form the small latent complex. As demonstrated in platelets, small latent complexes may be associated with additional high molecular weight proteins, the best characterized of which is the latent TGF-.beta. binding protein or LTBP (Kanzaki et al., 1990). (LTBP has been shown to be the 125-160 kDa subunit of the purified latent complex from platelets). Latent TGF-.beta. complexes that contain LTBP are also known as large latent complexes. In contrast to platelet LTBP, the LTBP produced by fibroblasts typically is a 190 kDa polypeptide. The smaller size of platelet LTBP may be due to proteolytic processing or alternative splicing (Kanzaki et al., 1990; Tsuji et al., 1990).
3. LAP and Latency
TGF-.beta. latency results in part from the non-covalent association of the propeptide dimer and the mature TGF-.beta. dimer (Pircher et al., 1984; Gentry et al., 1988; Wakefield et al., 1989). A cDNA for the TGF-.beta.1 precursor was expressed in Chinese Hamster Ovary (CHO) cells, which do not express LTBP (Gentry et al., 1988), and almost all TGF-.beta. activity recovered from the medium of transfected cells was latent. Use of deletion constructs has demonstrated that synthesis of biologically active TGF-.beta.1 can proceed only from the first ATG codon, implicating LAP in the proper assembly of the small latent complex in these cells. Taken together, these studies indicate that LAP is sufficient to achieve the latent state. More recent studies have shown that carbohydrate structures within LAP make an important contribution to the latent state. For example, treatment of the latent form of TGF-.beta.1 with endoglycosidase F led to activation of TGF-.beta. (Miyazono and Heldin, 1989). (The structure of the mature TGF-.beta. dimer was not affected by enzyme treatment). In particular, sialic acid residues seemed to be important, as treatment of the purified latent complex with sialidase was also able to activate TGF-.beta. from the latent state.
4. Modulation of Latency
Latent complexes must be dissociated to activate mature TGF-.beta., and dissociation is considered to be a critical step in governing TGF-.beta. effects (Twardzik et al., 1990; Sato et al., 1993). Dissociation by chemical treatment of the latent complex purified from platelets has been investigated (Miyazono et al., 1990). Incubation of the purified complex under conditions of varying pH revealed that TGF-.beta. activity was unmasked at values below pH 3.5 and above pH 12.5. Incubation of latent TGF-.beta. in 0.02% SDS or 8 M urea also effectively unmasked TGF-.beta. activity, but incubation in 5 M NaCl did not. Wakefield et al. (Wakefield et al., 1989) have reported that, after activation, TGF-.beta.1 and LAP reassociate in a time- and concentration-dependent manner under neutral, nondenaturing conditions. These results are consistent with the idea that the mature TGF-.beta. dimer is non-covalently associated with LAP.
Latent TGF-.beta. complexes are also dissociated by the action of certain enzymes. For example, latent TGF-.beta. is activated by plasmin, which disrupts the structure of the large latent complex (Lyons et al., 1988; Taipale et al., 1995). Similar data exist for other enzymes, e.g., cathepsin D, mast cell chymase, leukocyte elastase, and the glycosidases. Recently, osteoclast-derived cells were shown to be capable of activating latent TGF-.beta. in vitro (Oreffo et al., 1989). Osteoclast activation is of particular interest because of the hypothesis that TGF-.beta. serves as a link between bone turnover and formation during bone remodeling (Centrella et al., 1991). The mechanism of TGF-, activation by osteoclasts is not known at present, but it is reasonable to think that local alteration of pH due to action of proton pumps in the osteoclast plasma membrane or the release of osteoclast-derived proteases may be involved in the activation process. Related to these observations, activated macrophages (as might be found at a wound site or during tissue morphogenesis) secrete sialidase and other proteases (Pilatte et al., 1987), and they can lower the local pH to 4.0 (Silver et al., 1988), both of which could contribute to TGF-.beta. activation in vivo. As mentioned above, acidification weakens the non-covalent interaction between LAP and the mature TGF-.beta. dimer (Wakefield et al., 1989).